Diester and triester based low molecular weight, biodegradeable cationic lipids for oligonucleotide delivery

ABSTRACT

The instant invention provides for novel cationic lipids of Formula A that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with oligonucleotides. It is an object of the instant invention to provide a cationic lipid scaffold that demonstrates enhanced efficacy along with lower liver toxicity as a result of lower lipid levels in the liver. The present invention employs low molecular weight cationic lipids with one short lipid chain coupled with inclusion of hydrolysable functionality in the lipid chains to enhance the efficiency and tolerability of in vivo delivery of siRNA.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/198,271 filed Jun. 30, 2016, which application is a continuation ofU.S. application Ser. No. 14/395,267 filed Oct. 17, 2014, now U.S. Pat.No. 9,402,816 issued on Aug. 2, 2016, which is a 35 U.S.C. § 371National Phase Entry Application of International Application No.PCT/US2013/036682 filed Apr. 16, 2013, which designates the U.S., andwhich claims benefit under 35 U.S.C. § 119(e) of the U.S. ProvisionalApplication No. 61/635,494, filed Apr. 19, 2012, the content of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The sequence listing of the present application has been submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “23222WOPCTSEQ”, creation date of Jun. 29, 2016 and a size of11,409 bytes. The sequence listing submitted via EFS-Web is part of thespecification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to novel diester and triester cationiclipids that can be used in combination with other lipid components suchas cholesterol and PEG-lipids to form lipid nanoparticles witholigonucleotides, to facilitate the cellular uptake and endosomalescape, and to knockdown target mRNA both in vitro and in vivo.

Cationic lipids and the use of cationic lipids in lipid nanoparticlesfor the delivery of oligonucleotides, in particular siRNA and miRNA,have been previously disclosed. Lipid nanoparticles and use of lipidnanoparticles for the delivery of oligonucleotides, in particular siRNAand miRNA, have been previously disclosed. Oligonucleotides (includingsiRNA and miRNA) and the synthesis of oligonucleotides have beenpreviously disclosed. (See US patent applications: US 2006/0083780, US2006/0240554, US 2008/0020058, US 2009/0263407 and US 2009/0285881 andPCT patent applications: WO 2009/086558, WO2009/127060, WO2009/132131,WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405,WO2010/054406 and WO2011/153493). See also Semple S. C. et al., Rationaldesign of cationic lipids for siRNA delivery, Nature Biotechnology,2010, 28, 172-176.

Other cationic lipids are disclosed in the following patentapplications: US 2009/0263407, US 2009/0285881, US 2010/0055168, US2010/0055169, US 2010/0063135, US 2010/0076055, US 2010/0099738, US2010/0104629, WO2010/088537, WO2010/144740, US2010/0324120, U.S. Pat.No. 8,034,376, WO2011/143230, WO2011/000106, US2011/0117125,US2011/0256175, WO2011/141703, WO2011/141704 and WO2011/141705.

Traditional cationic lipids such as CLinDMA and DLinDMA have been usedfor siRNA delivery to the liver but suffer from non-optimal deliveryefficiency along with liver toxicity at higher doses. It is an object ofthe instant invention to provide a cationic lipid scaffold thatdemonstrates enhanced efficacy along with lower liver toxicity as aresult of lower lipid levels in the liver. The present invention employslow molecular weight cationic lipids with one short lipid chain coupledwith inclusion of hydrolysable functionality in the lipid chains toenhance the efficiency and tolerability of in vivo delivery of siRNA.

SUMMARY OF THE INVENTION

The instant invention provides for novel cationic lipids of Formula Athat can be used in combination with other lipid components such ascholesterol and PEG-lipids to form lipid nanoparticles witholigonucleotides. It is an object of the instant invention to provide acationic lipid scaffold that demonstrates enhanced efficacy along withlower liver toxicity as a result of lower lipid levels in the liver. Thepresent invention employs low molecular weight cationic lipids with oneshort lipid chain coupled with inclusion of hydrolysable functionalityin the lipid chains to enhance the efficiency and tolerability of invivo delivery of siRNA.

DETAILED DESCRIPTION OF THE INVENTION

The various aspects and embodiments of the invention are directed tonovel cationic lipids useful in lipid nanoparticles to deliveroligonucleotides, in particular, siRNA and miRNA, to any target gene.(See US patent applications: US 2006/0083780, US 2006/0240554, US2008/0020058, US 2009/0263407 and US 2009/0285881 and PCT patentapplications: WO 2009/086558, WO2009/127060, WO2009/132131,WO2010/042877, WO2010/054384, WO2010/054401, WO2010/054405,WO2010/054406 and WO2011/153493). See also Semple S. C. et al., Rationaldesign of cationic lipids for siRNA delivery, Nature Biotechnology,2010, 28, 172-176.

The cationic lipids of the instant invention are useful components in alipid nanoparticle for the delivery of oligonucleotides, specificallysiRNA and miRNA.

In a first embodiment of this invention, the cationic lipids areillustrated by Formula A:

wherein:

R¹ and R² are each independently selected from the group consisting ofH, (C₁-C₆)alkyl, heterocyclyl, and polyamine, wherein said alkyl,heterocyclyl and polyamine are optionally substituted with one to threeR′; or R¹ and R² can be taken together with the nitrogen to which theyare attached to form a monocyclic heterocycle with 4-7 membersoptionally containing, in addition to the nitrogen, one or twoadditional heteroatoms selected from N, O and S, said monocyclicheterocycle is optionally substituted with one to three R′;

R³ is selected from the group consisting of (C₄-C₂₀)alkyl and(C₄-C₂₀)alkenyl, said alkyl or alkenyl is optionally substituted withone to three R′;

R⁴ is selected from the group consisting of (C₁-C₁₆)alkyl and(C₂-C₁₆)alkenyl, said alkyl or alkenyl is optionally substituted withone to three R′;

R⁵ is selected from the group consisting of (C₄-C₈)alkyl and(C₄-C₈)alkenyl, said alkyl or alkenyl is optionally substituted with oneto three R′;

R⁶ is (C₁-C₂)alkyl, said alkyl is optionally substituted with one tothree R′;

Q¹ and Q² are each, independently, selected from the group consisting ofa bond, —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —S—S—, —C(R″)═N—,—N═C(R″)—, —C(R″)═N—O—, —O—N═C(R″)—, —C(O)(NR″)—, —N(R″)C(O)—,C(S)(NR″)—, —N(R″)C(O)—, —N(R″)C(O)N(R″)—, —OC(O)O—, OSi(R″)₂O—,—C(O)(CR″₂)C(O)O— and —OC(O)(CR″₂)C(O)—), with the proviso that wheneither Q¹ or Q² is a bond then the other is not a bond;

X is selected from the group consisting of —OC(O)—, —C(O)O—, —SC(O)—,—C(O)S—, —OC(S)—, —S—S—, —C(R″)═N—, —N═C(R″)—, —C(R″)═N—O—, —O—N═C(R″)—,—C(O)(NR″)—, —N(R″)C(O)—, C(S)(NR″)—, —N(R″)C(O)—, —N(R″)C(O)N(R″)—,—OC(O)O—, OSi(R″)₂O—, —C(O)(CR″₂)C(O)O— and —OC(O)(CR″₂)C(O)—),

each occurrence of R′ is independently selected from the groupconsisting of halogen, R″, OR″, SR″, CN, CO₂R″ and CON(R″)₂;

each occurrence of R″ is independently selected from the groupconsisting of H and (C₁-C₆)alkyl, wherein said alkyl is optionallysubstituted with halogen and OH; and

n is 1, 2, 3, 4 or 5;

or a pharmaceutically acceptable salt or stereoisomer thereof.

In a second embodiment, the invention features a compound having FormulaA as described above, wherein:

R¹ and R² are each methyl;

n is 3;

R³ is selected from (C₄-C₂₀)alkyl and (C₄-C₂₀)alkenyl, said alkyl oralkenyl is optionally substituted with one to three R′;

R⁴ is selected from (C₁-C₁₆)alkyl and (C₂-C₁₆)alkenyl, said alkyl oralkenyl is optionally substituted with one to three substituentsselected from R′;

R⁵ is selected from (C₄-C₈)alkyl and (C₄-C₈)alkenyl, said alkyl oralkenyl is optionally substituted with one to three R′;

R⁶ is (C₁-C₂)alkyl, said alkyl is optionally substituted with one tothree R′;

Q¹ and Q² are each, independently, a bond or —C(O)O—, with the provisothat when either Q¹ or Q² is a bond then the other is not a bond;

X is —C(O)O—; and

R′ is as defined above;

or a pharmaceutically acceptable salt or stereoisomer thereof.

Specific embodiments of the cationic lipids disclosed herein are:

-   methyl (9Z)-19-{[4-(dimethylamino)butanoyl]oxy}octacos-9-enoate    (Compound 1);-   methyl    8-[2-(9-{[4-(dimethylamino)butanoyl]oxy}octadecyl)cyclopropyl]octanoate    (Compound 2);-   methyl (9Z)-19-{[4-(dimethylamino)butanoyl]oxy}heptacos-9-enoate    (Compound 3);-   methyl (9Z)-19-{[4-(dimethylamino)butanoyl]oxy}hexacos-9-enoate    (Compound 4);-   methyl (9Z)-19-{[4-(dimethylamino)butanoyl]oxy}pentacos-9-enoate    (Compound 5);-   methyl (9Z)-21-{[4-(dimethylamino)butanoyl]oxy}triacont-9-enoate    (Compound 6);-   methyl (9Z)-21-{[4-(dimethylamino)butanoyl]oxy}nonacos-9-enoate    (Compound 7);-   methyl (9Z)-21-{[4-(dimethylamino)butanoyl]oxy}octacos-9-enoate    (Compound 8);-   methyl (9Z)-21-{[4-(dimethylamino)butanoyl]oxy}heptacos-9-enoate    (Compound 9);-   methyl (11Z)-19-{[4-(dimethylamino)butanoyl]oxy}octacos-11-enoate    (Compound 10);-   methyl (7Z)-19-{[4-(dimethylamino)butanoyl]oxy}octacos-7-enoate    (Compound 11);-   methyl    8-[2-(9-{[4-(dimethylamino)butanoyl]oxy}heptadecyl)cyclopropyl]octanoate    (Compound 12);-   methyl    8-[2-(9-{[4-(dimethylamino)butanoyl]oxy}hexadecyl)cyclopropyl]octanoate    (Compound 13);-   methyl    8-[2-(9-{[4-(dimethylamino)butanoyl]oxy}pentadecyl)cyclopropyl]octanoate    (Compound 14);-   methyl    8-[2-(11-{[4-(dimethylamino)butanoyl]oxy}icosyl)cyclopropyl]octanoate    (Compound 15);-   methyl    8-[2-(11-{[4-(dimethylamino)butanoyl]oxy}nonadecyl)cyclopropyl]octanoate    (Compound 16);-   methyl 8-{2-[11-(dimethylamino)octadecyl]cyclopropyl}octanoate    (Compound 17);-   methyl    8-[2-(11-{[4-(dimethylamino)butanoyl]oxy}octadecyl)cyclopropyl]octanoate    (Compound 18);-   methyl    10-[2-(7-{[4-(dimethylamino)butanoyl]oxy}hexadecyl)cyclopropyl]decanoate    (Compound 19);-   methyl    6-[2-(11-{[4-(dimethylamino)butanoyl]oxy}icosyl)cyclopropyl]hexanoate    (Compound 20);-   ethyl (7Z)-17-{[4-(dimethylamino)butanoyl]oxy}hexacos-7-enoate    (Compound 21);-   ethyl    6-[2-(9-{[4-(dimethylamino)butanoyl]oxy}octadecyl)cyclopropyl]hexanoate    (Compound 22);-   (2Z)-non-2-en-1-yl 10-{[4-(dimethylamino)butanoyl]oxy}nonadecanoate    (Compound 23);-   (2-hexylcyclopropyl)methyl    10-{[4-(dimethylamino)butanoyl]oxy}nonadecanoate (Compound 24);-   (2Z)-undec-2-en-1-yl    8-{[4-(dimethylamino)butanoyl]oxy}heptadecanoate (Compound 25);-   (2Z)-hept-2-en-1-yl 12-{[4-(dimethylamino)butanoyl]oxy}henicosanoate    (Compound 26);-   (2-octylcyclopropyl)methyl    8-{[4-(dimethylamino)butanoyl]oxy}heptadecanoate (Compound 27);-   (2-butylcyclopropyl)methyl    12-{[4-(dimethylamino)butanoyl]oxy}henicosanoate (Compound 28);-   methyl    (19Z,22Z)-9-{[4-(dimethylamino)butanoyl]oxy}octacosa-19,22-dienoate    (Compound 29);-   ethyl    (18Z,21Z)-8-{[4-(dimethylamino)butanoyl]oxy}heptacosa-18,21-dienoate    (Compound 30);-   methyl    9-{[4-(dimethylamino)butanoyl]oxy}-16-(2-octylcyclopropyl)hexadecanoate    (Compound 31);-   ethyl    8-{[4-(dimethylamino)butanoyl]oxy}-15-(2-octylcyclopropyl)pentadecanoate    (Compound 32);-   dimethyl    (9Z)-19-{[4-(dimethylamino)butanoyl]oxy}heptacos-9-enedioate    (Compound 33); and-   1-methyl 18-[(2Z)-non-2-en-1-yl]    9-{[4-(dimethylamino)butanoyl]oxy}octadecanedioate (Compound 34);    or a pharmaceutically acceptable salt or stereoisomer thereof.

In another embodiment, the cationic lipids disclosed are useful in thepreparation of lipid nanoparticles.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nanoparticle for the delivery of oligonucleotides.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nanoparticle for the delivery of siRNA and miRNA.

In another embodiment, the cationic lipids disclosed are usefulcomponents in a lipid nanoparticle for the delivery of siRNA.

The cationic lipids of the present invention may have asymmetriccenters, chiral axes, and chiral planes (as described in: E. L. Elieland S. H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons,New York, 1994, pages 1119-1190), and occur as racemates, racemicmixtures, and as individual diastereomers, with all possible isomers andmixtures thereof, including optical isomers, being included in thepresent invention. In addition, the cationic lipids disclosed herein mayexist as tautomers and both tautomeric forms are intended to beencompassed by the scope of the invention, even though only onetautomeric structure is depicted.

It is understood that substituents and substitution patterns on thecationic lipids of the instant invention can be selected by one ofordinary skill in the art to provide cationic lipids that are chemicallystable and that can be readily synthesized by techniques known in theart, as well as those methods set forth below, from readily availablestarting materials. If a substituent is itself substituted with morethan one group, it is understood that these multiple groups may be onthe same carbon or on different carbons, so long as a stable structureresults.

It is understood that one or more Si atoms can be incorporated into thecationic lipids of the instant invention by one of ordinary skill in theart to provide cationic lipids that are chemically stable and that canbe readily synthesized by techniques known in the art from readilyavailable starting materials.

In the compounds of Formula A, the atoms may exhibit their naturalisotopic abundances, or one or more of the atoms may be artificiallyenriched in a particular isotope having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberpredominantly found in nature. The present invention is meant to includeall suitable isotopic variations of the compounds of Formula A. Forexample, different isotopic forms of hydrogen (H) include protium (1H)and deuterium (²H). Protium is the predominant hydrogen isotope found innature. Enriching for deuterium may afford certain therapeuticadvantages, such as increasing in vivo half-life or reducing dosagerequirements, or may provide a compound useful as a standard forcharacterization of biological samples. Isotopically-enriched compoundswithin Formula A can be prepared without undue experimentation byconventional techniques well known to those skilled in the art or byprocesses analogous to those described in the Scheme and Examples hereinusing appropriate isotopically-enriched reagents and/or intermediates.

As used herein, “alkyl” means a straight chain, cyclic or branchedsaturated aliphatic hydrocarbon having the specified number of carbonatoms.

As used herein, “alkenyl” means a straight chain, cyclic or branchedunsaturated aliphatic hydrocarbon having the specified number of carbonatoms including but not limited to diene, triene and tetraeneunsaturated aliphatic hydrocarbons.

Examples of a cyclic “alkyl” or “alkenyl include:

As used herein, “heterocyclyl” or “heterocycle” means a 4- to10-membered aromatic or nonaromatic heterocycle containing from 1 to 4heteroatoms selected from the group consisting of O, N and S, andincludes bicyclic groups. “Heterocyclyl” therefore includes, thefollowing: benzoimidazolyl, benzofuranyl, benzofurazanyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl,indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl,pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl,thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl,hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl,thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, andN-oxides thereof all of which are optionally substituted with one tothree substituents selected from R″.

As used herein, “polyamine” means compounds having two or more aminogroups. Examples include putrescine, cadaverine, spermidine, andspermine.

As used herein, “halogen” means F, Cl, Br or I.

In an embodiment of Formula A, R¹ and R² are each independently selectedfrom H and (C₁-C₆)alkyl, wherein said alkyl is optionally substitutedwith one to three R′; or R¹ and R² can be taken together with thenitrogen to which they are attached to form a monocyclic heterocyclewith 4-7 members optionally containing, in addition to the nitrogen, oneor two additional heteroatoms selected from N, O and S, said monocyclicheterocycle is optionally substituted with one to three R′.

In an embodiment of Formula A, R¹ and R² are each independently selectedfrom H, methyl, ethyl and propyl, wherein said methyl, ethyl and propylare optionally substituted with one to three R′; or R¹ and R² can betaken together with the nitrogen to which they are attached to form amonocyclic heterocycle with 4-7 members optionally containing, inaddition to the nitrogen, one or two additional heteroatoms selectedfrom N, O and S, said monocyclic heterocycle is optionally substitutedwith one to three substituents selected from R′.

In an embodiment of Formula A, R¹ and R² are each independently selectedfrom H, methyl, ethyl and propyl.

In an embodiment of Formula A, R¹ and R² are each methyl.

In an embodiment of Formula A, R³ is selected from (C₄-C₂₀)alkyl and(C₄-C₂₀)alkenyl.

In an embodiment of Formula A, R³ is (C₁₄-C₁₈)alkenyl.

In an embodiment of Formula A, R³ is C₁₆alkenyl.

In an embodiment of Formula A, R³ is C₁₄ alkenyl.

In an embodiment of Formula A, R³ is (C₆-C₉)alkyl.

In an embodiment of Formula A, R³ is C₇alkyl.

In an embodiment of Formula A, R⁴ is (C₁-C₁₆)alkyl or (C₂-C₁₆)alkenyl.

In an embodiment of Formula A, R⁴ is (C₄-C₁₀)alkenyl.

In an embodiment of Formula A, R⁴ is C₉alkenyl.

In an embodiment of Formula A, R⁴ is (C₁-C₄)alkyl.

In an embodiment of Formula A, R⁴ is (C₁-C₂)alkyl.

In an embodiment of Formula A, R⁴ is ethyl.

In an embodiment of Formula A, R⁴ is methyl.

In an embodiment of Formula A, R³ is C₇alkyl and R⁴ is C₉alkenyl.

In an embodiment of Formula A, R³ is C₁₄alkenyl and R⁴ is ethyl.

In an embodiment of Formula A, R³ is C₁₆alkenyl and R⁴ is methyl.

In an embodiment of Formula A, R⁵ is (C₄-C₈)alkyl or (C₄-C₈)alkenyl.

In an embodiment of Formula A, R⁵ is (C₄-C₈)alkyl.

In an embodiment of Formula A, R⁵ is (C₆-C₈)alkyl.

In an embodiment of Formula A, R⁵ is C₈alkyl.

In an embodiment of Formula A, R⁶ is methyl or ethyl.

In an embodiment of Formula A, R⁶ is ethyl.

In an embodiment of Formula A, R⁶ is methyl.

In an embodiment of Formula A, R⁵ is C₈alkyl and R⁴ is ethyl.

In an embodiment of Formula A, R⁵ is C₈alkyl and R⁴ is methyl.

In an embodiment of Formula A, R⁵ is C₇alkyl and R⁴ is ethyl.

In an embodiment of Formula A, R⁵ is C₇alkyl and R⁴ is methyl.

In an embodiment of Formula A, Q¹ and Q² are each, independentlyselected from a bond, —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—,—S—S—, —C(R″)═N—, —N═C(R″)—, —C(R″)═N—O—, —O—N═C(R″)—, —C(O)(NR″)—,—N(R″)C(O)—, C(S)(NR″)—, —N(R″)C(O)—, —N(R″)C(O)N(R″)—, —OC(O)O—,OSi(R″)₂O—, —C(O)(CR″₂)C(O)O— and —OC(O)(CR″₂)C(O)—, with the provisothat when either Q¹ or Q² is a bond then the other is not a bond.

In an embodiment of Formula A, Q¹ and Q² are each, independently a bondor —C(O)O—, with the proviso that when either Q¹ or Q² is a bond thenthe other is not a bond.

In an embodiment of Formula A, X is selected from —OC(O)—, —C(O)O—,—SC(O)—, —C(O)S—, —OC(S)—, —S—S—, —C(R″)═N—, —N═C(R″)—, —C(R″)═N—O—,—O—N═C(R″)—, —C(O)(NR″)—, —N(R″)C(O)—, C(S)(NR″)—, —N(R″)C(O)—,—N(R″)C(O)N(R″)—, —OC(O)O—, OSi(R″)₂O—, —C(O)(CR″₂)C(O)O— and—OC(O)(CR″₂)C(O)—.

In an embodiment of Formula A, X is —C(O)O—

In an embodiment of Formula A, R′ is R″.

In an embodiment of Formula A, R″ is independently selected from H,methyl, ethyl and propyl, wherein said methyl, ethyl and propyl areoptionally substituted with one or more halogen and OH.

In an embodiment of Formula A, R″ is independently selected from H,methyl, ethyl and propyl.

In an embodiment of Formula A, n is 1, 2, 3, 4 or 5.

In an embodiment of Formula A, n is 3.

In an embodiment of Formula A, “heterocyclyl” is pyrolidine, piperidine,morpholine, imidazole or piperazine.

In an embodiment of Formula A, “monocyclic heterocyclyl” is pyrolidine,piperidine, morpholine, imidazole or piperazine.

In an embodiment of Formula A, “polyamine” is putrescine, cadaverine,spermidine or spermine.

In an embodiment, “alkyl” is a straight chain saturated aliphatichydrocarbon having the specified number of carbon atoms.

In an embodiment, “alkenyl” is a straight chain unsaturated aliphatichydrocarbon having the specified number of carbon atoms.

Included in the instant invention is the free form of cationic lipids ofFormula A, as well as the pharmaceutically acceptable salts andstereoisomers thereof. Some of the isolated specific cationic lipidsexemplified herein are the protonated salts of amine cationic lipids.The term “free form” refers to the amine cationic lipids in non-saltform. The encompassed pharmaceutically acceptable salts not only includethe isolated salts exemplified for the specific cationic lipidsdescribed herein, but also all the typical pharmaceutically acceptablesalts of the free form of cationic lipids of Formula A. The free form ofthe specific salt cationic lipids described may be isolated usingtechniques known in the art. For example, the free form may beregenerated by treating the salt with a suitable dilute aqueous basesolution such as dilute aqueous NaOH, potassium carbonate, ammonia andsodium bicarbonate. The free forms may differ from their respective saltforms somewhat in certain physical properties, such as solubility inpolar solvents, but the acid and base salts are otherwisepharmaceutically equivalent to their respective free forms for purposesof the invention.

The pharmaceutically acceptable salts of the instant cationic lipids canbe synthesized from the cationic lipids of this invention which containa basic or acidic moiety by conventional chemical methods. Generally,the salts of the basic cationic lipids are prepared either by ionexchange chromatography or by reacting the free base with stoichiometricamounts or with an excess of the desired salt-forming inorganic ororganic acid in a suitable solvent or various combinations of solvents.Similarly, the salts of the acidic compounds are formed by reactionswith the appropriate inorganic or organic base.

Thus, pharmaceutically acceptable salts of the cationic lipids of thisinvention include the conventional non-toxic salts of the cationiclipids of this invention as formed by reacting a basic instant cationiclipids with an inorganic or organic acid. For example, conventionalnon-toxic salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like, as well as salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,trifluoroacetic (TFA) and the like.

When the cationic lipids of the present invention are acidic, suitable“pharmaceutically acceptable salts” refers to salts prepared formpharmaceutically acceptable non-toxic bases including inorganic basesand organic bases. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc and the like. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as arginine, betainecaffeine, choline, N,N′-dibenzylethylenediamine, diethylamin,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, thiobromine, triethylamine, trimethylaminetripropylamine, tromethamine and the like.

The preparation of the pharmaceutically acceptable salts described aboveand other typical pharmaceutically acceptable salts is more fullydescribed by Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci.,1977:66:1-19.

It will also be noted that the cationic lipids of the present inventionare potentially internal salts or zwitterions, since under physiologicalconditions a deprotonated acidic moiety in the compound, such as acarboxyl group, may be anionic, and this electronic charge might then bebalanced off internally against the cationic charge of a protonated oralkylated basic moiety, such as a quaternary nitrogen atom.

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be further illustrative of the invention and not limitativeof the reasonable scope thereof. The reagents utilized in synthesizingthe cationic lipids are either commercially available or readilyprepared by one of ordinary skill in the art.

Synthesis of the novel cationic lipids is a linear process starting fromlipid aldehyde (i). Addition of a lipid based Grignard reagent cangenerate secondary alcohol (ii). This alcohol is protected as its silylether (iii) and the olefin is dihydroxylated with osmium tetroxide togive diol (iv). The diol is oxidatively cleaved with sodium periodate toprovide aldehyde (v). The aldehyde is converted to the carboxylic acidcontaining olefin (vi) by a Wittig olenfination. The acid is convertedto the ester (vii) in situ, followed by silyl ether deprotection to givealcohol (viii). The alcohol is then coupled to give final compounds ix.Cyclopropanation then provides additional lipids x.

Synthesis of ester containing lipids (xiii) is achieved by oxidation ofaldehyde v to carboxylic acid xi, followed by ester formation.Conversion to xiii is completed in a manner analogous to that describedin General Scheme 1.

Synthesis of ester containing lipids xxi is a linear sequence beginningwith carboxylic acid xiv. The acid is converted to the Weinreb amide xvfollowed by Grignard addition to give ketone xvi. The alcohol isdeprotected, oxidized and esterified to give ester xix. Ketone xix isconverted to final compound xxi via reduction and ester coupling.

Synthesis of diester amines is accomplished as outlined in GeneralScheme 4. Alkene xxii is dihydroxylated and oxidatively cleaved to givealdehyde xxiv. The aldehyde is converted to the carboxylic acidcontaining alkene via a Wittig olefination. The resulting acid xxv isconverted to its corresponding ester in situ. The silyl ether isdeprotected and the alcohol oxidized to give carboxylic acid xxvii. Thisis esterified to give ketone intermediate xxviii. This ketone is carriedon to final compound xxix as outlined in General Schemes 1 and 3 above.

Synthesis of bis-ester compounds (xxxii) is achieved by oxidation ofaldehyde (xxiv) followed by esterification to give ester xxxi.Conversion to the final compounds is achieved in a manner analogous tothat described above in General Schemes 1-4.

Methyl (9Z)-19-{[4-(dimethylamino)butanoyl]oxy}octacos-9-enoate(Compound 1)

Oleyl aldehyde (a) in THF is cooled to 0° C. and treated withnonylmagnesium bromide. The reaction is warmed to room temperature andquenched with aqueous bicarbonate solution upon completion. The reactionmixture is partitioned between water and hexanes, the organics driedover sodium sulfate, filtered and evaporated in vacuo to give crudealcohol (b). The crude product is purified by flash columnchromatography.

Alcohol (b) is taken up in dichloromethane and treated with triethylamine and DMAP. To this solution is added TBDPSCl in a single portion atambient temperature. The reaction is quenched with aqueous bicarbonatesolution upon completion. The reaction mixture is partitioned betweenwater and hexanes, the organics dried over sodium sulfate, filtered andevaporated in vacuo to give crude silyl ether (c). The crude product ispurified by flash column chromatography.

Silyl ether (c) is taken up in a mixture of tert-butanol, THF and waterand treated with osmium tetroxide and NMO. The reaction is quenched withaqueous bicarbonate solution upon completion. The reaction mixture ispartitioned between water and hexanes, the organics dried over sodiumsulfate, filtered and evaporated in vacuo to give crude diol (d). Thecrude product is purified by flash column chromatography.

Diol (d) is taken up in a mixture of THF, dichloromethane, methanol andwater and treated with sodium periodate. The reaction is quenched withaqueous bicarbonate solution upon completion. The reaction mixture ispartitioned between water and hexanes, the organics dried over sodiumsulfate, filtered and evaporated in vacuo to give crude aldehyde (e).The crude product is purified by flash column chromatography.

Ylide precursor triphenylphosphinium bromide is taken up in THF andtreated with HMPA and lithium hexamethyldisilazide to generate theylide. To this solution is added aldehyde (e). Upon reaction completion,the solution is treated with sodium bicarbonate and dimethylsulfate. Thereaction is quenched with aqueous bicarbonate solution upon completion.The reaction mixture is partitioned between water and hexanes, theorganics dried over sodium sulfate, filtered and evaporated in vacuo togive crude ester (f). The crude product is purified by flash columnchromatography.

Ester (f) is taken up in THF and treated with TBAF. The reaction isquenched with aqueous bicarbonate solution upon completion. The reactionmixture is partitioned between water and hexanes, the organics driedover sodium sulfate, filtered and evaporated in vacuo to give crudealcohol (g). The crude product is purified by flash columnchromatography.

Alcohol (g) and 4-(dimethylamino)butanoic acid are taken up indichloromethane and treated with EDC, DMAP and DIEA at ambienttemperature. The reaction is quenched with aqueous bicarbonate solutionupon completion. The reaction mixture is partitioned between water andhexanes, the organics dried over sodium sulfate, filtered and evaporatedin vacuo to give crude Compound 1. The crude product is purified byflash column chromatography.

Methyl8-[2-(9-{[4-(dimethylamino)butanoyl]oxy}octadecyl)cyclopropyl]octanoate(Compound 2)

A solution of diethylzinc in dichloromethane is cooled to −1° C. andtreated dropwise with TFA. After 30 minutes, diiodomethane is added andthe resulting solution aged for 30 minutes in an ice bath. To thissolution is added olefin Compound 1 and the resulting solution is warmedslowly to ambient temperature. The reaction is quenched with aqueousbicarbonate solution upon completion. The reaction mixture ispartitioned between water and hexanes, the organics dried over sodiumsulfate, filtered and evaporated in vacuo to give crude cyclopropaneCompound 2. The crude product is purified by flash columnchromatography.

Compounds 3-22 are novel cationic lipids and are prepared according tothe General Scheme 1 described above.

Compound Structure Name 3

methyl (9Z)-19-{[4- (dimethylamino)butanoyl] oxy}heptacos-9- enoate 4

methyl (9Z)-19-{[4- (dimethylamino)butanoyl oxy}hexacos-9- enoate 5

methyl (9Z)-19-{[4- (dimethylamino)butanoyl] oxy}pentacos-9- enoate 6

methyl (9Z)-21-{[4- (dimethylamino)butanoyl] oxy}triacont-9-enoate 7

methyl (9Z)-21-{[4- (dimethylamino)butanoyl] oxy}nonacos-9- enoate 8

methyl (9Z)-21-{[4- (dimethylamino)butanoyl] oxy}octacos-9-enoate 9

methyl (9Z)-21-{[4- (dimethylamino)butanoyl] oxy}heptacos-9- enoate 10

methyl (11Z)-19-{[4- (dimethylamino)butanoyl] oxy}octacos-11- enoate 11

methyl (7Z)-19-{[4- (dimethylamino)butanoyl] oxy}octacos-7-enoate 12

methyl 8-[2-(9-{[4- (dimethylamino)butanoyl] oxy}heptadecyl)cyclopropyl]octanoate 13

methyl 8-[2-(9-{[4- (dimethylamino)butanoyl] oxy}hexadecyl)cyclopropyl]octanoate 14

methyl 8-[2-(9-{[4- (dimethylamino)butanoyl] oxy}pentadecyl)cyclopropyl]octanoate 15

methyl 8-[2-11-{[4- (dimethylamino)butanoyl] oxy}icosyl)cyclopropyl]octanoate 16

methyl 8-[2-(11-{[4- (dimethylamino)butanoyl] oxy}nonadecyl)cyclopropyl]octanoate 17

methyl 8-{2-[11- (dimethylamino)octadecyl] cyclopropyl}octanoate 18

methyl 8-[2-(11-{[4- (dimethylamino)butanoyl] oxy}octadecyl)cyclopropyl]octanoate 19

methyl 10-[2-(7-{[4- (dimethylamino)butanoyl] oxy}hexadecyl)cyclopropyl]decanoate 20

methyl 6-[2-(11-{[4- (dimethylamino)butanoyl] oxy}icosyl)cyclopropyl]hexanoate 21

ethyl (7Z)-17-{[4- (dimethylamino)butanoyl] oxy}hexacos-7- enoate 22

ethyl 6-[2-(9-{[4- (dimethylamino)butanoyl] oxy}octadecyl)cyclopropyl]hexanoate

(2Z)-non-2-en-1-yl 10-{[4-(dimethylamino)butanoyl]oxy}nonadecanoate(Compound 23)

A solution of aldehyde (e) in DMF is treated with Oxone at ambienttemperature. The reaction is quenched with ammonium chloride solutionand partitioned between hexanes and water upon completion. The organicsare dried over sodium sulfate, filtered and evaporated in vacuo to givecrude acid (h). This material is purified by flash chromatography.

A solution of acid (h) and C9-alcohol in DMF is treated with EDCI anddiisopropylethylamine. The reaction is quenched with ammonium chloridesolution and partitioned between hexanes and water upon completion. Theorganics are dried over sodium sulfate, filtered and evaporated in vacuoto give crude acid (i). This material is purified by flashchromatography.

Conversion of silyl ether (i) to Compound 23 is carried out in a manneranalogous to that described for Compound 1 above

(2-hexylcyclopropyl)methyl10-{[4-(dimethylamino)butanoyl]oxy}nonadecanoate (Compound 24)

Compound 24 is prepared in a manner analogous to that described abovefor compound 23 employing the cyclopropanation chemistry as describedfor compound 2 above.

Compounds 25-28 are novel cationic lipids and are prepared according toGeneral Schemes 1-2 above.

Compound Structure Name 25

(2Z)-undec-2-en-1-yl 8-{[4- (dimethylamino) butanoyl]oxy} heptadecanoate26

(2Z)-hept-2-en-1-yl 12-{[4- (dimethylamino) butanoyl]oxy} heptadecanoate27

(2- octylcyclopropyl) methyl 8-{[4- (dimethylamino) butanoyl]oxy}heptadecanoate 28

(2- butylcyclopropyl) methyl 12-{[4- (dimethylamino) butanoyl]oxy}henicosanoate

Methyl(19Z,22Z)-9-{[4-(dimethylamino)butanoyl]oxy}octacosa-19,22-dienoate(Compound 29)

11,14-Eicosadienoic acid, (11Z,14Z)— (50 g, 162 mmol),N,O-Dimethylhydroxylamine hydrochloride (31.6 g, 324 mmol), HOAt (44.1g, 324 mmol), Et₃N (45.2 mL, 324 mmol), and EDC (62.1 g, 324 mmol) weremixed in DCM (810 mL) and stirred overnight at ambient temperature.Reaction was then washed 5×700 mL water, then washed 1×600 mL 1 M NaOH,dried with sodium sulfate, filtered through celite and evaporated toobtain 53.06 g (93%) 11,14-eicosadienamide, N-methoxy-N-methyl-,(11Z,14Z) as a clear golden oil. ¹H NMR (400 MHz, CDCl₃) δ 5.35 (m, 4H),3.68 (s, 3H), 3.18 (s, 3H), 2.77 (m, 2H), 2.41 (t, J=7 Hz, 2H), 2.05 (m,4H), 1.63 (m, 2H), 1.40-1.26 (m, 18H), 0.89 (t, J=7 Hz, 3H).

A solution of alkyl bromide in THF is treated with magnesium turnings togenerate the Grignard reagent. A separate solution of Weinreb amide (1)in THF is treated with this resulting Grignard solution at ambienttemperature. The reaction is quenched with ammonium chloride solutionand partitioned between hexanes and water upon completion. The organicsare dried over sodium sulfate, filtered and evaporated in vacuo to givecrude ketone (m). This material is purified by flash chromatography.

A solution of silyl ether (m) in THF is treated with TBAF. The reactionis quenched with ammonium chloride solution and partitioned betweenhexanes and water upon completion. The organics are dried over sodiumsulfate, filtered and evaporated in vacuo to give crude alcohol (n).This material is purified by flash chromatography.

A solution of alcohol (n) in DMF is treated with pyridinium dichromateat 0° C. The solution is warmed to ambient temperature. The reaction isquenched with water and partitioned between hexanes and water uponcompletion. The organics are dried over sodium sulfate, filtered andevaporated in vacuo to give crude acid (o). This material is purified byflash chromatography.

A solution of acid (o) in THF is treated with sodium bicarbonate anddimethylsulfate. The solution is warmed to ambient temperature. Thereaction is quenched with sodium bicarbonate solution and partitionedbetween hexanes and water upon completion. The organics are dried oversodium sulfate, filtered and evaporated in vacuo to give crudeketo-ester (p). This material is purified by flash chromatography.

Ketone (p) is taken up in methanol and treated with sodium borohydride.The reaction is quenched with sodium bicarbonate and partitioned betweenwater/hexanes. The organics are dried over sodium sulfate, filtered andevaporated in vacuo to give crude alcohol (q). This is purified by flashchromatography.

Alcohol (q) and 4-(dimethylamino)butanoic acid are taken up indichloromethane and treated with EDC, DMAP and DIEA at ambienttemperature. The reaction is quenched with aqueous bicarbonate solutionupon completion. The reaction mixture is partitioned between water andhexanes, the organics dried over sodium sulfate, filtered and evaporatedin vacuo to give crude lipid Compound 29. The crude product is purifiedby flash column chromatography.

Compounds 30-32 are novel cationic lipids and are prepared according toGeneral Schemes 1-3 above.

Compound Structure Name 30

ethyl (18Z,21Z)-8-{[4- (dimethylamino)butanoyl] oxy}heptacosa-18,21-dienoate 31

methyl 9-{[4- (dimethylamino)butanoyl] oxy}-16-(2- octylcyclopropyl)hexadecanoate 32

ethyl 8-{[4- (dimethylamino)butanoyl] oxy}-15-(2- octycylopropyl)pentadecanoate

Dimethyl (9Z)-19-{[4-(dimethylamino)butanoyl]oxy}heptacos-9-enedioate(Compound 33)

solution of alkyl bromide in THF is treated with magnesium turnings andaged to generate the Grignard reagent. A separate solution of Weinrebamide is treated with the Grignard reagent. The reaction is quenchedwith sodium bicarbonate solution and partitioned between hexanes andwater upon completion. The organics are dried over sodium sulfate,filtered and evaporated in vacuo to give crude ketone (r). This materialis purified by flash chromatography.

A solution of ketone in THF, tert-butanol and water is treated withosmium tetroxide and NMO. The reaction is quenched with sodiumbicarbonate solution and partitioned between hexanes and water uponcompletion. The organics are dried over sodium sulfate, filtered andevaporated in vacuo to give crude diol (s). This material is purified byflash chromatography.

A solution of diol (s) is taken up in THF, dichloromethane, methanol andwater and treated with sodium periodate. The reaction is quenched withsodium bicarbonate solution and partitioned between hexanes and waterupon completion. The organics are dried over sodium sulfate, filteredand evaporated in vacuo to give crude aldehyde (t). This material ispurified by flash chromatography.

Ylide precursor triphenylphosphinium bromide is taken up in THF andtreated with HMPA and lithium hexamethyldisilazide to generate theylide. To this solution is added aldehyde (t). Upon reaction completion,the solution is treated with sodium bicarbonate and dimethylsulfate. Thereaction is quenched with aqueous bicarbonate solution upon completion.The reaction mixture is partitioned between water and hexanes, theorganics dried over sodium sulfate, filtered and evaporated in vacuo togive crude ester (u). The crude product is purified by flash columnchromatography.

A solution of silyl ether (u) in THF is treated with TBAF. The reactionis quenched with aqueous bicarbonate solution upon completion. Thereaction mixture is partitioned between water and hexanes, the organicsdried over sodium sulfate, filtered and evaporated in vacuo to givecrude alcohol. The crude product is purified by flash columnchromatography.

A solution of alcohol in DMF is treated with pyridinium dichromate. Thereaction is quenched with water upon completion. The reaction mixture ispartitioned between water and hexanes, the organics dried over sodiumsulfate, filtered and evaporated in vacuo to give crude acid (v). Thecrude product is purified by flash column chromatography.

A solution of acid (v) in THF is treated with sodium bicarbonate anddimethyl sulfate. The reaction is quenched with aqueous bicarbonatesolution upon completion. The reaction mixture is partitioned betweenwater and hexanes, the organics dried over sodium sulfate, filtered andevaporated in vacuo to give crude diester (w). The crude product ispurified by flash column chromatography.

Ketone (w) is converted to amine Compound 33 in a manner analogous tothat described for Compound 29.

Diesters similar to Compound 33 are prepared wherein modifications tothe structure are similar to those outlined in the tables above, i.e.varying lipid chain lengths, methyl and ethyl esters, inclusion ofcylcopropanes, modifying position of unsaturation or cyclopropaneincorporation, and all possible combinations of above.

1-Methyl 18-[(2Z)-non-2-en-1-yl9-{4-(dimethylamino)butanoyl]oxy}octadecanedioate (Compound 34)

A solution of aldehyde (t) in DMF is treated with pyridinium dichromate.The reaction is quenched with aqueous bicarbonate solution uponcompletion. The reaction mixture is partitioned between water andhexanes, the organics dried over sodium sulfate, filtered and evaporatedin vacuo to give crude acid (x). The crude product is purified by flashcolumn chromatography.

A solution of acid (x) in dichloromethane is treated with C9 alcohol andEDCI and diisopropylethylamine. The reaction is quenched with aqueousbicarbonate solution upon completion. The reaction mixture ispartitioned between water and hexanes, the organics dried over sodiumsulfate, filtered and evaporated in vacuo to give crude keto ester (y).The crude product is purified by flash column chromatography.

Ketone (y) is converted to amine Compound 34 in a manner analogous tothat described for Compound 33 above.

Diesters similar to Compound 34 are prepared wherein modifications tothe structure are similar to those outlined in the tables above, i.e.varying lipid chain lengths, methyl and ethyl esters, inclusion ofcylcopropanes, modifying position of unsaturation or cyclopropaneincorporation, and all possible combinations of above.

LNP COMPOSITIONS

The following lipid nanoparticle compositions (LNPs) of the instantinvention are useful for the delivery of oligonucleotides, specificallysiRNA and miRNA:

Cationic Lipid/Cholesterol/PEG-DMG 56.6/38/5.4;

Cationic Lipid/Cholesterol/PEG-DMG 60/38/2;

Cationic Lipid/Cholesterol/PEG-DMG 67.3/29/3.7;

Cationic Lipid/Cholesterol/PEG-DMG 49.3/47/3.7;

Cationic Lipid/Cholesterol/PEG-DMG 50.3/44.3/5.4;

Cationic Lipid/Cholesterol/PEG-C-DMA/DSPC 40/48/2/10;

Cationic Lipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10; and

Cationic Lipid/Cholesterol/PEG-DMG/DSPC 58/30/2/10.

LNP Process Description:

The Lipid Nano-Particles (LNP) is prepared by an impinging jet process.The particles are formed by mixing lipids dissolved in alcohol withsiRNA dissolved in a citrate buffer. The mixing ratio of lipids to siRNAare targeted at 45-55% lipid and 65-45% siRNA. The lipid solution cancontain a novel cationic lipid of the instant invention, a helper lipid(cholesterol), PEG (e.g. PEG-C-DMA, PEG-DMG) lipid, and DSPC at aconcentration of 5-15 mg/mL with a target of 9-12 mg/mL in an alcohol(for example ethanol). The ratio of the lipids can have a mole percentrange of 25-98 for the cationic lipid with a target of 35-65, the helperlipid can have a mole percent range from 0-75 with a target of 30-50,the PEG lipid can have a mole percent range from 1-15 with a target of1-6, and the DSPC can have a mole percent range of 0-15 with a target of0-12. The siRNA solution can contain one or more siRNA sequences at aconcentration range from 0.3 to 1.0 mg/mL with a target of 0.3-0.9 mg/mLin a sodium citrate buffered salt solution with pH in the range of3.5-5. The two liquids are heated to a temperature in the range of15-40° C., targeting 30-40° C., and then mixed in an impinging jet mixerinstantly forming the LNP. The teeID can have a range from 0.25 to 1.0mm and a total flow rate from 10-600 mL/min. The combination of flowrate and tubing ID can have the effect of controlling the particle sizeof the LNPs between 30 and 200 nm. The solution can then be mixed with abuffered solution at a higher pH with a mixing ratio in the range of 1:1to 1:3 vol:vol but targeting 1:2 vol:vol. This buffered solution is at atemperature in the range of 15-40° C., targeting 30-40° C. The mixedLNPs are held from 30 minutes to 2 hrs prior to an anion exchangefiltration step. The temperature during incubating is in the range of15-40° C., targeting 30-40° C. After incubating the solution is filteredthrough a 0.8 um filter containing an anion exchange separation step.This process can use tubing IDs ranging from 1 mm ID to 5 mm ID and aflow rate from 10 to 2000 mL/min. The LNPs are concentrated anddiafiltered via an ultrafiltration process where the alcohol is removedand the citrate buffer is exchanged for the final buffer solution suchas phosphate buffered saline. The ultrafiltration process can use atangential flow filtration format (TFF). This process can use a membranenominal molecular weight cutoff range from 30-500 KD. The membraneformat is hollow fiber or flat sheet cassette. The TFF processes withthe proper molecular weight cutoff can retain the LNP in the retentateand the filtrate or permeate contains the alcohol; citrate buffer; finalbuffer wastes. The TFF process is a multiple step process with aninitial concentration to a siRNA concentration of 1-3 mg/mL. Followingconcentration, the LNPs solution is diafiltered against the final bufferfor 10-20 volumes to remove the alcohol and perform buffer exchange. Thematerial can then be concentrated an additional 1-3 fold. The finalsteps of the LNP process are to sterile filter the concentrated LNPsolution and vial the product.

Analytical Procedure:

1) siRNA Concentration

The siRNA duplex concentrations are determined by Strong Anion-ExchangeHigh-Performance Liquid Chromatography (SAX-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a 2996 PDAdetector. The LNPs, otherwise referred to as RNAi Delivery Vehicles(RDVs), are treated with 0.5% Triton X-100 to free total siRNA andanalyzed by SAX separation using a Dionex BioLC DNAPac PA 200 (4×250 mm)column with UV detection at 254 nm. Mobile phase is composed of A: 25 mMNaClO₄, 10 mM Tris, 20% EtOH, pH 7.0 and B: 250 mM NaClO₄, 10 mM Tris,20% EtOH, pH 7.0 with liner gradient from 0-15 min and flow rate of 1ml/min. The siRNA amount is determined by comparing to the siRNAstandard curve.

2) Encapsulation Rate

Fluorescence reagent SYBR Gold is employed for RNA quantitation tomonitor the encapsulation rate of RDVs. RDVs with or without TritonX-100 are used to determine the free siRNA and total siRNA amount. Theassay is performed using a SpectraMax M5e microplate spectrophotometerfrom Molecular Devices (Sunnyvale, Calif.). Samples are excited at 485nm and fluorescence emission is measured at 530 nm. The siRNA amount isdetermined by comparing to the siRNA standard curve.Encapsulation rate=(1−free siRNA/total siRNA)×100%3) Particle Size and Polydispersity

RDVs containing 1 μg siRNA are diluted to a final volume of 3 ml with1×PBS. The particle size and polydispersity of the samples is measuredby a dynamic light scattering method using ZetaPALS instrument(Brookhaven Instruments Corporation, Holtsville, N.Y.). The scatteredintensity is measured with He—Ne laser at 25° C. with a scattering angleof 90°.

4) Zeta Potential Analysis

RDVs containing 1 μg siRNA are diluted to a final volume of 2 ml with 1mM Tris buffer (pH 7.4). Electrophoretic mobility of samples isdetermined using ZetaPALS instrument (Brookhaven InstrumentsCorporation, Holtsville, N.Y.) with electrode and He—Ne laser as a lightsource. The Smoluchowski limit is assumed in the calculation of zetapotentials.

5) Lipid Analysis

Individual lipid concentrations is determined by Reverse PhaseHigh-Performance Liquid Chromatography (RP-HPLC) using Waters 2695Alliance system (Water Corporation, Milford Mass.) with a Corona chargedaerosol detector (CAD) (ESA Biosciences, Inc, Chelmsford, Mass.).Individual lipids in RDVs are analyzed using an Agilent Zorbax SB-C18(50×4.6 mm, 1.8 μm particle size) column with CAD at 60° C. The mobilephase is composed of A: 0.1% TFA in H₂O and B: 0.1% TFA in IPA. Thegradient can change from 60% mobile phase A and 40% mobile phase B fromtime 0 to 40% mobile phase A and 60% mobile phase B at 1.00 min; 40%mobile phase A and 60% mobile phase B from 1.00 to 5.00 min; 40% mobilephase A and 60% mobile phase B from 5.00 min to 25% mobile phase A and75% mobile phase B at 10.00 min; 25% mobile phase A and 75% mobile phaseB from 10.00 min to 5% mobile phase A and 95% mobile phase B at 15.00min; and 5% mobile phase A and 95% mobile phase B from 15.00 to 60%mobile phase A and 40% mobile phase B at 20.00 min with flow rate of 1ml/min. The individual lipid concentration is determined by comparing tothe standard curve with all the lipid components in the RDVs with aquadratic curve fit. The molar percentage of each lipid is calculatedbased on its molecular weight.

Utilizing the above described LNP process, specific LNPs with thefollowing ratios are identified:

Nominal Composition:

Cationic Lipid/Cholesterol/PEG-DMG 60/38/2

Cationic Lipid/Cholesterol/PEG-DMG/DSPC 58/30/2/10

Luc siRNA

(SEQ. ID. NO.: 1) 5′-iB-A U AAGG CU A U GAAGAGA U ATT-iB 3′(SEQ. ID. NO.: 2) 3′-UUUAUUCCGAUACUUCUC UAU-5′ AUGC-RiboseiB-Inverted deoxy abasic UC-2′ Flouro AGT-2′ Deoxy AGU-2′ OCH₃Nominal CompositionCationic Lipid/Cholesterol/PEG-DMG 60/38/2Cationic Lipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10Cationic Lipid/Cholesterol/PEG-DMG/DSPC 58/30/2/10ApoB siRNA

(SEQ ID NO.: 3) 5′-iB-CUUUAACAAUUCCUGAAAUTsT-iB-3′ (SEQ ID NO.: 4)3′-UsUGAAAUUGUUAAGGACUs UsUsA-5′ AUGC-Ribose iB-Inverted deoxy abasicUC-2′ Flouro AGT-2′ Deoxy AGU-2′ OCH₃ UsA-phortherothioate linkagebeta-catenin siRNA (SEQ ID NO.: 5) 5′-iB-CUGUUGGAUUGAUUCGAAAUsU-iB-3′′(SEQ ID NO.: 6) 3′-UsUG ACA A CCUAA C UAAGCUUU-5′ AUGC-RiboseiB-Inverted deoxy abasic UC-2′ Fluoro AGT-2′ Deoxy AGU-2′ OCH₃UsA-phophorothioate linkage (SEQ ID NO.: 7) 5′-iB-A C GACUA GUUC AGUU GCUUUsU-iB-3′ (SEQ ID NO.: 8) 3′-UsUUGCUGAUCAAGUCA ACGAA-5′ AUGC-RiboseiB-Inverted deoxy abasic UC-2′ Fluoro AGT-2′ Deoxy AGU-2′ OCH₃UsA-phophorothioate linkage (SEQ ID NO.: 9) 5′-iB-A C GACUA GUUC AGUU GCUUUU-iB-3′ (SEQ ID NO.: 10) 3′-UUUGCUGAUCAAGUC A ACGAA-5′ AUGC-RiboseiB-Inverted deoxy abasic UC-2′ Fluoro AGT-2′ Deoxy AGU-2′ OCH₃UsA-phophorothioate linkage

Oligonucleotide synthesis is well known in the art. (See US patentapplications: US 2006/0083780, US 2006/0240554, US 2008/0020058, US2009/0263407 and US 2009/0285881 and PCT patent applications: WO2009/086558, WO2009/127060, WO2009/132131, WO2010/042877, WO2010/054384,WO2010/054401, WO2010/054405 and WO2010/054406). The siRNAs disclosedand utilized in the Examples are synthesized via standard solid phaseprocedures.

Example 1

Mouse In Vivo Evaluation of Efficacy

LNPs utilizing Compounds 1-34, in the nominal compositions describedimmediately above, are evaluated for in vivo efficacy. The siRNA cantarget the mRNA transcript for the firefly (Photinus pyralis) luciferasegene (Accession # M15077). The primary sequence and chemicalmodification pattern of the luciferase siRNA is displayed above. The invivo luciferase model employs a transgenic mouse in which the fireflyluciferase coding sequence is present in all cells.ROSA26-LoxP-Stop-LoxP-Luc (LSL-Luc) transgenic mice licensed from theDana Farber Cancer Institute are induced to express the Luciferase geneby first removing the LSL sequence with a recombinant Ad-Cre virus(Vector Biolabs). Due to the organo-tropic nature of the virus,expression is limited to the liver when delivered via tail veininjection. Luciferase expression levels in liver are quantitated bymeasuring light output, using an IVIS imager (Xenogen) followingadministration of the luciferin substrate (Caliper Life Sciences).Pre-dose luminescence levels is measured prior to administration of theRDVs. Luciferin in PBS (15 mg/mL) is intraperitoneally (IP) injected ina volume of 150 μL. After a four minute incubation period mice areanesthetized with isoflurane and placed in the IVIS imager. The RDVs(containing siRNA) in PBS vehicle are tail vein injected in a volume of0.2 mL. Final dose levels can range from 0.1 to 0.5 mg/kg siRNA. PBSvehicle alone is dosed as a control. Mice are imaged 48 hours post doseusing the method described above. Changes in luciferin light outputdirectly correlate with luciferase mRNA levels and represent an indirectmeasure of luciferase siRNA activity. In vivo efficacy results areexpressed as % inhibition of luminescence relative to pre-doseluminescence levels.

Example 2

In Vitro ApoE Binding Assay

LNPs are incubated at 37° C. in 90% rhesus serum at a final LNPconcentration of 4 ug/mL. Incubation is for 20 minutes with orbitalrotation. After incubation, the samples are diluted 1:20 in PBS and 100μL of each diluted sample is aliquoted to wells of an anti-PEG antibodycoated 96-well plate (Life Diagnostics Cat. No. P-0001PL. Afterincubation at room temperature for 1 hour, the plate is washed 5× with300 uL PBS. After washing, 50 uL of 0.2% Triton X-100 is added to eachwell and the plate incubated at 37° C. for 10 minutes, followed byshaking on a plate shaker for 1 minute at 750 rpm. Samples are frozenprior to performing the ApoE ELISA and stem loop PCR analysis ofsamples.

An ApoE ELISA assay is performed to quantitate ApoE bound to the LNPsafter incubation in rhesus serum. Anti-ApoE antibody (Milipore, Cat No.AB947) is diluted 1:1000 in PBS and 100 μL of diluted antibody is addedto each well of a polystyrene high binding plate. The plate withantibody is incubated overnight at 4° C., after which the plate iswashed 2× with 200 μL of PBS. Next, 200 μL of buffer containing 1% BSAand 0.05% Tween-20 in PBS (Incubation Buffer) is added to each wellfollowed by incubation at room temperature for 1 hour. Plates are washed5× with PBS containing 0.05% Tween-20. Frozen Triton lysis test samplesare thawed and diluted 1:6 with incubation buffer and 100 μL of testsample is aliquoted to wells of the ApoE antibody plate. Incubation isfor 1 hour at room temperature followed by a 5× wash with PBS containing0.05% Tween-20. After washing, 100 μL of biotinylated anti-ApoE antibody(Mabtech, Cat. ANo. E887-biotin), diluted 1:500 in incubation buffer, isadded to each well and incubated for 1 hour at room temperature,followed by a 5× wash with 0.05% Tween-20 in PBS. 100 μL per well, ofStreptavidin-HPR (Thermo, Cat. No. TS-125-HR), is then added andincubated for 1 hour at room temperature. After washing 5× with 0.05%Tween-20 in PBS, 100 μL of TMB Substrate (Thermo, Cat. No. 34028) isadded to each well, followed by incubation at room temperature for 20minutes in the dark. The colorimetric reaction is stopped with 100 μL ofTMB Stop Solution (KPL, Cat. No. 50-85-04) and absorbance at 450 nm isdetermined. An ApoE standard curve is prepared by diluting rhesusRecombinant ApoE in incubation buffer with 0.03% Triton X-100 withconcentrations ranging from 100 ng/mL to 0.78 ng/mL. ApoE standards areevaluated in the ELISA in parallel to the test samples. A rhesus serumonly (no LNP) control is utilized to obtain a background subtraction fornon-LNP dependent ApoE signal in the ELISA.

Stem Loop RT-PCR Protocol

To normalize to the ApoE bound to the amount of LNP bound to theanti-PEG antibody plate, the amount of siRNA retained in the anti-PEGantibody well is quantitated by stem-loop PCR and related to the numberof siRNAs encapsulated per LNP, to give an approximate measure of totalLNP particles bound per well.

Preparation of the Spiked Standard Curve Samples:

The standard curve is prepared using the molecular weight of the siRNA(13693 g/mol for ApoB 17063) to calculate the copy number. The highstandard should contain 10¹¹ copies per 3 μl. A 10-fold serial dilutionis performed across a row of an assay plate until the lowest standardcontains 10² copies per 3.1. One could dilute 0.2% Triton X-100 1:80 inwater and pipette 20 μL of the diluted Triton X-100 into 10 wells of a96 well plate. 30 μL of the serial diluted standard curve and mix isadded to each well of the plate. 10 μL of the spiked standard curve isused in the reverse transcription reaction.

Stem-Loop RT-PCR—Test Samples and Standard Curve:

Triton lysates from the PEG antibody plate capture is diluted 1 to 2000in nuclease free water. 10 μL of ‘RT-Primer Mix’ (Applied Biosystem'sTaqMan MicroRNA Reverse Transcription Kit Cat. No. 4366596) is added toeach well of a 96-well Micro-Amp QPCR plate (ABI Cat# N801-0560).

RT Primer Mix Components μL/rxn Final conc. ApoB RT-primer (10 uM) 0.6200 nM 10× buffer 2 Water 7.4

ApoB RT primer sequence:  (SEQ. ID. NO.: 11) 5′GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC CTTTAACA3′

10 μL of each test sample (diluted 1 to 2000) or spiked standard curve(above) is aliquoted into the 96-well plate. The plate is covered with amat (ABI Cat. No. N801-0550), to minimize evaporation. The plate isbriefly centrifuged at 800 rpm for 1 minute. Next, the plate is run on athermocycler using the following cycling parameters:

Cycling: 94° C. 10 minutes  75° C. 2 minutes 60° C. 3 minutes 50° C. 3minutes 40° C. 3 minutes 30° C. 3 minutes  4° C. hold

Next, 10 μL of ‘RT Mix’ is added to each well (Applied Biosystem'sTaqMan MicroRNA Reverse Transcription Kit Cat. No. 4366596)

RT Mix Components μL/rxn 100 mM dNTP 0.3 10x RT buffer 1 Rnase Inhibitor0.38 Multiscribe RT enzyme 1 Water 7.32

The RT cycling reaction is composed of 10 μL test sample, 10 μL of RTprimer mix and 10 μL of RT Mix components for a total volume of 30 μL.The final concentration of the RT-primer in the total 30 μL total RT mixis 200 nM. The plate is then sealed with the same plate mat, brieflycentrifuged at 800 rpm for 1 minute, then run on the thermocycler usingthe following cycling parameters:

Cycling: 16° C. 30 minutes 42° C. 30 minutes 85° C.  5 minutes  4° C.hold

Next, 15 μL of Fast Enzyme/primer-probe mix is added to each well of anew Fast 96-well plate (Applied Biosystem's TaqMan Fast Universal PCRMaster Mix, Cat. No. 4352042)

ApoB PCR Master Mix Components μL/rxn Final Conc. Fast Enyzme Mix (2×stock) 10 forward primer (100 uM) 0.18 900 nM reverse primer (100 uM)0.18 900 nM probe (10 uM) 0.05 250 nM Water 4.59

ApoB primers and probe sequence: (SEQ. ID. NO.: 12)17063DC F3 GGCGCGAAATTTCAGGAATTGT (SEQ. ID. NO.: 13)17063DC Pr2 CACTGGATACGACCTTTAACA (SEQ. ID. NO.: 14)Universal R2 AGTGCAGGGTCCGAG

5 μL of each RT reaction is added to the Fast Enzyme Mix plate. Theplate is centrifuged for 1 minute at 1000 rpm and the QPCR analysis isperformed on an ABI7900 with Fast Block. Cycling parameters is: 1cycle—95° C. for 20 seconds, followed by 40 Cycles—95° C. for 1 seconds,60° C. for 20 seconds.

The QPCR result is utilized to calculate the siRNA concentration in thePEG antibody capture plate Triton lysates. Based on an estimate of 500siRNA per LNP particle, the number of LNPs retained in each well of theanti-PEG antibody plate can be calculated. Using the ApoE concentrationper well, as determined by the ApoE ELISA and the number of LNPparticles per well, an approximate ApoE molecules bound per LNP particlecan be calculated.

Example 3

Heparin Sepharose HI-TRAP™ Binding Assay

Lipid nanoparticles (LNP) with neutral surface charge are not retainedafter injection onto heparin sepharose with 1× Dulbecco's phosphatebuffered saline (DPBS) as the running buffer but elute in the columnvoid volume. Serum apolipoprotein E (ApoE) exhibits high affinitybinding with heparin sulfate and it can be shown that LNPs bind toheparin sepharose to an extent dependent on their intrinsic ability tobind ApoE (depending on both lipid nanoparticle composition and ApoEconcentration) after incubation with purified and/or recombinant humanApoE or serum samples. Lipid nanoparticles with surface bound ApoE bindto heparin sepharose with high affinity can be eluted only at high salt(1M NaCl).

A heparin sepharose binding assay is developed to assess serum ApoEbinding to lipid nanoparticles based on the high affinity interactionthat ApoE-LNP complexes exhibit toward heparin sepharose.

Incubations

Lipid nanoparticles are incubated at 37° C. for 20 min at a final siRNAconcentration of 50 μg/mL with various concentrations of either purifiedor recombinant human apolipoprotein E or 0.1-50% rat/mouse/rhesusmonkey/human serum in 1×Dulbecco's phosphate buffered saline (DPBS).After incubation with ApoE or serum LNP samples are diluted 10-foldusing 1×DPBS and analyzed by heparin sepharose chromatography. Peak areaof retained LNP (after subtraction of appropriate blank signals) iscompared to total peak area of LNP control without ApoE and/or serumincubation to determine the percentage of the LNP which undergoes shiftto high affinity heparin interaction after incubation with ApoE/serum.

Heparin Sepharose HI-TRAP™ Chromatographic Conditions

A heparin sepharose HI-TRAP™ chromatography column (GE Healthcare; 1 mLbed volume) is equilibrated with either 1× or 2× Dulbecco's PBS; thehigher 2× salt concentration is used for LNPs with higher intrinsicretention on heparin sepharose (presumably due to higher positivesurface charge).

Mobile Phase A: 1× or 2×DPBS

Mobile Phase B: 1M NaCl in 10 mM sodium phosphate buffer, pH 7.0

100% A delivered isocratically for 10 min followed by step gradient to100% B; hold for additional 10 min; step gradient back to 100% A andreequilibrate for additional 10 min prior to injection of next sample

Flow rate: 1 mL/min

Sample injection volume: 50 μL.

Detection: UV @260 nm

Example 4

Rat In Vivo Evaluation of Efficacy and Toxicity

LNPs utilizing compounds in the nominal compositions described above,are evaluated for in vivo efficacy and increases in alanine aminotransferase and aspartate amino transferase in Sprague-Dawley(Crl:CD(SD) female rats (Charles River Labs). The siRNA targets the mRNAtranscript for the ApoB gene (Accession # NM 019287). The primarysequence and chemical modification pattern of the ApoB siRNA isdisplayed above. The RDVs (containing siRNA) in PBS vehicle are tailvein injected in a volume of 1 to 1.5 mL. Infusion rate is approximately3 ml/min. Five rats are used in each dosing group. After LNPadministration, rats are placed in cages with normal diet and waterpresent. Six hours post dose, food is removed from the cages. Animalnecropsy is performed 24 hours after LNP dosing. Rats are anesthetizedunder isoflurane for 5 minutes, then maintained under anesthesia byplacing them in nose cones continuing the delivery of isoflurane untilex-sanguination is completed. Blood is collected from the vena cavausing a 23 gauge butterfly venipuncture set and aliquoted to serumseparator vacutainers for serum chemistry analysis. Punches of theexcised caudate liver lobe is taken and placed in RNALater (Ambion) formRNA analysis. Preserved liver tissue is homogenized and total RNAisolated using a Qiagen bead mill and the Qiagen miRNA-Easy RNAisolation kit following the manufacturer's instructions. Liver ApoB mRNAlevels are determined by quantitative RT-PCR. Message is amplified frompurified RNA utilizing a rat ApoB commercial probe set (AppliedBiosystems Cat # RN01499054_m1). The PCR reaction is performed on an ABI7500 instrument with a 96-well Fast Block. The ApoB mRNA level isnormalized to the housekeeping PPIB (NM 011149) mRNA. PPIB mRNA levelsare determined by RT-PCR using a commercial probe set (Applied BiosytemsCat. No. Mm00478295_m1). Results are expressed as a ratio of ApoBmRNA/PPIB mRNA. All mRNA data is expressed relative to the PBS controldose. Serum ALT and AST analysis is performed on the Siemens Advia 1800Clinical Chemistry Analyzer utilizing the Siemens alanineaminotransferase (Cat#03039631) and aspartate aminotransferase(Cat#03039631) reagents.

Example 5

Determination of Cationic Lipid Levels in Rat/Monkey Liver

Liver tissue is weighed into 20-ml vials and homogenized in 9 v/w ofwater using a GenoGrinder 2000 (OPS Diagnostics, 1600 strokes/min, 5min). A 50 μL aliquot of each tissue homogenate is mixed with 300 μL ofextraction/protein precipitating solvent (50/50 acetonitrile/methanolcontaining 500 nM internal standard) and the plate is centrifuged tosediment precipitated protein. A volume of 200 μL of each supernatant isthen transferred to separate wells of a 96-well plate and 10 μl sampleswere directly analyzed by LC/MS-MS.

Standards are prepared by spiking known amounts of a methanol stocksolution of compound into untreated rat liver homogenate (9 volwater/weight liver). Aliquots (50 μL) each standard/liver homogenate ismixed with 300 μL of extraction/protein precipitating solvent (50/50acetonitrile/methanol containing 500 nM internal standard) and the plateis centrifuged to sediment precipitated protein. A volume of 200 μL ofeach supernatant is transferred to separate wells of a 96-well plate and10 μl of each standard is directly analyzed by LC/MS-MS.

Absolute quantification versus standards prepared and extracted fromliver homogenate is performed using an Aria LX-2 HPLC system (ThermoScientific) coupled to an API 4000 triple quadrupole mass spectrometer(Applied Biosystems). For each run, a total of 10 μL sample is injectedonto a BDS Hypersil C8 HPLC column (Thermo, 50×2 mm, 3 m) at ambienttemperature.

Mobile Phase A:

95% H2O/5% methanol/10 mM ammonium formate/0.1% formic acid Mobile PhaseB: 40% methanol/60% n-propanol/10 mM ammonium formate/0.1% formic acidThe flow rate is 0.5 mL/min and gradient elution profile is as follows:hold at 80% A for 0.25 min, linear ramp to 100% B over 1.6 min, hold at100% B for 2.5 min, then return and hold at 80% A for 1.75 min. Totalrun time is 5.8 min. API 4000 source parameters is CAD: 4, CUR: 15, GS1:65, GS2: 35, IS: 4000, TEM: 550, CXP: 15, DP: 60, EP: 10.

Example 6

Rhesus Monkey In Vivo Evaluation of ApoB Efficacy

LNPs utilizing compounds in the nominal compositions described above,are evaluated for in vivo efficacy in male or female Macaca mulatta(rhesus) monkeys. The siRNA targets the mRNA transcript for the ApoBgene (Accession # XM 001097404). The primary sequence and chemicalmodification pattern of the ApoB siRNA is displayed above. The RDVs(containing siRNA) in PBS vehicle are administered by intravenousinjection in the saphenous vein at an injection rate of 20 mL/minute toa dose level of 0.25 mg/kilogram siRNA. The injection volumes are from1.9 to 2.1 mL/kilogram and monkeys can range in weight from 2.5 to 4.5kilograms. The RDV or PBS control is administered to three monkeys. Atmultiple days post dose, 1 mL blood samples are drawn from the femoralartery for serum chemistry analysis. Monkeys are fasted overnight priorto blood draws. As a measure of efficacy, LDL-C is monitored as adownstream surrogate marker of ApoB mRNA reduction.

Example 7

Rhesus Monkey In Vivo Evaluation of β-Catenin Efficacy

On study day −7 predose liver biopsy samples (˜0.5-1 gram/sample) arecollected from male rhesus monkeys by laparoscopic surgical resection(resection of one biopsy sample from outer edge of one randomly selectedliver lobe per monkey). A 5 mm tissue punch is used to sample threenon-adjacent ˜50 mg samples from each predose biopsy. Samples arepreserved in RNAlater™ (Ambion) for later CTNNB1 mRNA analysis.

On study day 0 monkeys are administered suspensions of the lipidnanoparticle (LNP) test articles in phosphate buffered saline (0.05-0.1mg siRNA/mL) via single-dose intravenous bolus injection at target dosesof 0.67, 1.34 or 3.34 mg siRNA/m². For dosing purposes, body surfacearea (m²) is estimated from body weight according to the establishedallometric scaling relationship given below (1):BSA (m²)=0.11*BW (in kg)^(0.65)

On study days 2 and 7, at 48 hours and 168 hrs post LNP administration,liver biopsy samples (˜0.5-1 gram/sample) are collected from monkeys bylaparoscopic surgical resection (2 separate randomly selected liverlobes were resected per monkey). A 5 mm tissue punch is used to samplethree non-adjacent ˜50 mg samples per each 48 hr and 168 hr surgicalbiopsy sample. Samples are preserved in RNAlater™ (Ambion) for laterCTNNB1 mRNA analysis.

CTNNB1 mRNA levels are measured by relative quantitative RT-PCR using aprimer/probe set validated for CTNNB1 and normalized against mRNA levelsof peptidylprolyl isomerase B (also known as PPIB or cyclophilin B) andRNA levels of 18S ribosomal RNA (18S rRNA). Change in CTNNB1 mRNA liverexpression are measured as the difference in PCR threshold cycle number(ΔΔCt) between post-dose samples and each corresponding monkey's predoseliver samples.

Calculation of CTNNB1 mRNA knockdown (with respect to pretreatmentlevels) is calculated from ΔΔCt using the following relationship:mRNA(% knockdown)=100−(100/2^(−ΔΔCt))(1) FDA Guidance Document: “Guidance for Industry: Estimating theMaximum Safe Starting Dose in Initial Clinical Trials for Therapeuticsin Adult Healthy Volunteers” July 2005, US Department of Health andHuman Services, Food and Drug Administration—Center for Drug Evaluationand Research (CDER)

Example 8

Rhesus Monkey In Vivo Evaluation of ALT Increases

Alanine aminotransferase (ALT) is measured in serum that is harvestedfrom clotted monkey whole blood after centrifugation. A Roche ModularSystem automated chemistry analyzer measures the enzymatic activity ofALT in the serum by using International Federation of Clinical Chemistrystandardized procedures and reagents. The analyzer's computer usesabsorbance measurements to calculated ALT activity in the sample ascompared to a standard curve. The ALT activity is reported inInternational Units per Liter (IU/L).

What is claimed is:
 1. A cationic lipid of Formula A:

wherein: R¹ and R² are each independently selected from the groupconsisting of H, (C₁-C₆)alkyl, heterocyclyl, and polyamine, wherein saidalkyl, heterocyclyl and polyamine are optionally substituted with one tothree R′; or R¹ and R² can be taken together with the nitrogen to whichthey are attached to form a monocyclic heterocycle with 4-7 membersoptionally containing, in addition to the nitrogen, one or twoadditional heteroatoms selected from N, O and S, said monocyclicheterocycle is optionally substituted with one to three R′; R³ isselected from the group consisting of (C₄-C₂₀)alkyl and (C₄-C₂₀)alkenyl,said alkyl or alkenyl is optionally substituted with one to three R′; R⁴is selected from the group consisting of (C₁-C₁₆)alkyl and(C₂-C₁₆)alkenyl, said alkyl or alkenyl is optionally substituted withone to three R′; R⁵ is selected from the group consisting of(C₄-C₈)alkyl and (C₄-C₈)alkenyl, said alkyl or alkenyl is optionallysubstituted with one to three R′; R⁶ is (C₁-C₂)alkyl optionallysubstituted with one to three R′; Q¹ and Q² are each, independently,selected from the group consisting of a bond, —OC(O)—, —C(O)O—, —SC(O)—,—C(O)S—, —OC(S)—, —S—S—, —C(R″)═N—, —N═C(R″)—, —C(R″)═N—O—, —O—N═C(R″)—,—C(O)(NR″)—, —N(R″)C(O)—, C(S)(NR″)—, —N(R″)C(O)—, —N(R″)C(O)N(R″)—,—OC(O)O—, OSi(R″)₂O—, —C(O)(CR″₂)C(O)O— and —OC(O)(CR″₂)C(O)—), with theproviso that when either Q¹ or Q² is a bond then the other is not abond; X is selected from the group consisting of —OC(O)—, —C(O)O—,—SC(O)—, —C(O)S—, —OC(S)—, —S—S—, —C(R″)═N—, —N═C(R″)—, —C(R″)═N—O—,—O—N═C(R″)—, —C(O)(NR″)—, —N(R″)C(O)—, C(S)(NR″)—, —N(R″)C(O)—,—N(R″)C(O)N(R″)—, —OC(O)O—, OSi(R″)₂O—, —C(O)(CR″₂)C(O)O— and—OC(O)(CR″₂)C(O)—), each occurrence of R′ is independently selected fromthe group consisting of halogen, R″, OR″, SR″, CN, CO₂R″ or CON(R″)₂;each occurrence of R″ is independently selected from the groupconsisting of H and (C₁-C₆)alkyl, wherein said alkyl is optionallysubstituted with halogen and OH; and n is 1, 2, 3, 4 or 5; or apharmaceutically acceptable salt or stereoisomer thereof; with theproviso that at least one of R³-Q¹-R⁴ or R⁵-Q²-R⁶ is


2. A cationic lipid of Formula A according to claim 1, wherein: R¹ andR² are each methyl; n is 3; R³ is selected from the group consisting of(C₄-C₂₀)alkyl and (C₄-C₂₀)alkenyl, said alkyl or alkenyl is optionallysubstituted with one to three R′; R⁴ is selected from the groupconsisting of (C₁-C₁₆)alkyl and (C₂-C₁₆)alkenyl, said alkyl or alkenylis optionally substituted with one to three R′; R⁵ is selected from thegroup consisting of (C₄-C₈)alkyl and (C₄-C₈)alkenyl, said alkyl oralkenyl is optionally substituted with one to three R′; R⁶ is(C₁-C₂)alkyl optionally substituted with one to three R′; Q¹ and Q² areeach, independently, a bond or —C(O)O—, with the proviso that wheneither Q¹ or Q² is a bond then the other is not a bond; and X is—C(O)O—; or a pharmaceutically acceptable salt or stereoisomer thereof;with the proviso that at least one of R³-Q¹-R⁴ or R⁵-Q²-R⁶ is


3. A lipid nanoparticle comprising a cationic lipid of claim
 1. 4. Thelipid nanoparticle of claim 3, wherein the lipid nanoparticle furthercomprises an oligonucleotide.
 5. The lipid nanoparticle of claim 4,wherein the oligonucleotide is an siRNA or miRNA.
 6. The lipidnanoparticle of claim 5, wherein the oligonucleotide is an siRNA.
 7. Thelipid nanoparticle of claim 3, wherein the lipid nanoparticle furthercomprises cholesterol and PEG-DMG.
 8. The lipid nanoparticle of claim 3,wherein the lipid nanoparticle further comprises cholesterol, PEG-DMGand DSPC.
 9. The lipid nanoparticle of claim 3, wherein the lipidnanoparticle further comprises cholesterol, PEG-C-DMA and DSPC.